The synthesis of several oligosaccharides, in yields up to 65%, has been achieved by utilizing the transglycosylation activity of Agrobacterium sp. beta-glucosidase (EC 3.2.1.21). The regioselectivity of the glycosylation reaction was investigated using p-nitrophenyl beta-D-galactopyranoside, beta-D-mannopyranosyl fluoride and D-glucal as glycosyl donors and a series of phenyl 1-thio-, benzyl 1-thio-, and benzyl beta-D-glycopyranosides as glycosyl accepters. The isolated products were determined to contain a mixture of beta-(1 –> 3) and/or beta-(1 –> 4),glycosidic linkages for all glycosyl accepters, except for beta-D-galacto-configured accepters where a mixture of beta-(1 –> 3) and beta-(1 –> 6) linkages was observed. The wide range of glycosyl donors and accepters utilized by the enzyme in transglycosylation experiments demonstrates the versatility of this system for the aqueous-phase synthesis of oligosaccharides. (C) 1998 Elsevier Science Ltd.